Introduction to Applications of SNPS
Accumulation of genetic changes affecting cell cycle control, cell differentiation, apoptosis, and DNA replication and repair lead to carcinogenesis (Bishop, J. M., “Molecular Themes In Oncogenesis,” Cell, 64(2):235-48 (1991)). DNA alterations include large deletions which inactivate tumor supressor genes, amplification to increase expression of oncogenes, and most commonly single nucleotide mutations or polymorphisms which impair gene expression or gene function or predispose an individual to further genomic instability (Table 1).
TABLE 1Genetic Alterations Commonly Found in the Human GenomeType of AlterationPossible Causes of AlterationPossible Consequences of AlterationDetection of AlterationSingle nucleotideInherited variationSilent does not alter functionDNA sequencingpolymorphismMethylationMissense: alters gene functionSSCP, DGGE, CDGE(SNP)CarcinogensNonsense: truncates geneProtein truncationDefective repair genesMismatch cleavageMicrosatelliteDefective DNA repair genesFrameshift truncates geneMicrosatellite Analysisinstability (MIN)CarcinogensLarge deletionsDefective DNA repair genesLoss of gene functionLoss of heterozygosityDefective DNA replication genesCGHIllegitimate recombinationSNP analysisDouble strand breakDNA amplificationsDefective DNA repair genesOverexpression of geneCompetitive PCRDefective DNA replication genesCGHIllegitimate recombinationSNP analysisOthers:Defective methylase genesGene silencing or overexpression,Endonuclease digestionMethylation,Double strand breakcreation of chimeric proteinPCR, FISHTranslocationRapid detection of germline mutations in individuals at risk and accurate characterization of genetic changes in individual tumors would provide opportunities to improve early detection, prevention, prognosis, and specific treatment. However, genetic detection poses the problem of identifying a predisposing polymorphism in the germline or an index mutation in a pre-malignant lesion or early cancer that may be present at many potential sites in many genes. Furthermore, quantification of allele copy number is necessary to detect gene amplification and deletion. Therefore, technologies are urgently needed that can rapidly detect mutation, allele deletion, and allele amplification in multiple genes. Single nucleotide polymorphisms (“SNP”s) are potentially powerful genetic markers for early detection, diagnosis, and staging of human cancers.
Identification of DNA sequence polymorphisms is the cornerstone of modern genome mapping. Initially, maps were created using RFLP markers (Botstein, D., et al., “Construction Of A Genetic Linkage Map In Man Using Restriction Fragment Length Polymorphisms,” Amer. J. Hum. Genet., 32:314-331 (1980)), and later by the more polymorphic dinucleotide repeat sequences (Weber, J. L. et al., “Abundant Class Of Human DNA Polymorphisms Which Can Be Typed Using The Polymerase Chain Reaction,” Amer. J. Hum. Genet., 44:388-396 (1989) and Reed, P. W., et al., “Chromosome-Specific Microsatellite Sets For Fluorescence-Based, Semi-Automated Genome Mapping,” Nat Genet, 7(3): 390-5 (1994)). Such sequence polymorphisms may also be used to detect inactivation of tumor suppressor genes via LOH and activation of oncogenes via amplification. These genomic changes are currently being analyzed using conventional Southern hybridizations, competitive PCR, real-time PCR, microsatellite marker analysis, and comparative genome hybridization (CGH) (Ried, T., et al., “Comparative Genomic Hybridization Reveals A Specific Pattern Of Chromosomal Gains And Losses During The Genesis Of Colorectal Tumors,” Genes, Chromosomes & Cancer, 15(4):234-45 (1996), Kallioniemi, et al., “ERBB2 Amplification In Breast Cancer Analyzed By Fluorescence In Situ Hybridization,” Proc Natl Acad Sci USA, 89(12):5321-5 (1992), Kallioniemi, et al., “Comparative Genomic Hybridization: A Rapid New Method For Detecting And Mapping DNA Amplification In Tumors,” Semin Cancer Biol, 4(1):41-6 (1993), Kallioniemi, et al., “Detection And Mapping Of Amplified DNA Sequences In Breast Cancer By Comparative Genomic Hybridization,” Proc Natl Acad Sci USA, 91(6):2156-60 (1994), Kallioniemi, et al., “Identification Of Gains And Losses Of DNA Sequences In Primary Bladder Cancer By Comparative Genomic Hybridization,” Genes Chromosom Cancer, 12(3):213-9 (1995), Schwab, M., et al., “Amplified DNA With Limited Homology To Myc Cellular Oncogene Is Shared By Human Neuroblastoma Cell Lines And A Neuroblastoma Tumour,” Nature, 305(5931):245-8 (1983), Solomon, E., et al., “Chromosome 5 Allele Loss In Human Colorectal Carcinomas,” Nature, 328(6131):616-9 (1987), Law, D. J., et al., “Concerted Nonsyntenic Allelic Loss In Human Colorectal Carcinoma,” Science, 241(4868):961-5 (1988), Frye, R. A., et al., “Detection Of Amplified Oncogenes By Differential Polymerase Chain Reaction,” Oncogene, 4(9):1153-7 (1989), Neubauer, A., et al., “Analysis Of Gene Amplification In Archival Tissue By Differential Polymerase Chain Reaction,” Oncogene, 7(5):1019-25 (1992), Chiang, P. W., et al., “Use Of A Fluorescent-PCR Reaction To Detect Genomic Sequence Copy Number And Transcriptional Abundance,” Genome Research, 6(10):1013-26 (1996), Heid, C. A., et al., “Real Time Quantitative PCR,” Genome Research, 6(10):986-94 (1996), Lee, H. H., et al., “Rapid Detection Of Trisomy 21 By Homologous Gene Quantitative PCR (HGQ-PCR),” Human Genetics, 99(3):364-7 (1997), Boland, C. R., et al., “Microallelotyping Defines The Sequence And Tempo Of Allelic Losses At Tumour Suppressor Gene Loci During Colorectal Cancer Progression,” Nature Medicine, 1(9):902-9 (1995), Cawkwell, L., et al., “Frequency Of Allele Loss Of DCC, p53, RBI, WT1, NF1, NM23 And APC/MCC In Colorectal Cancer Assayed By Fluorescent Multiplex Polymerase Chain Reaction,” Br J Cancer, 70(5):813-8 (1994), and Hampton, G. M., et al., “Simultaneous Assessment Of Loss Of Heterozygosity At Multiple Microsatellite Loci Using Semi-Automated Fluorescence-Based Detection: Subregional Mapping Of Chromosome 4 In Cervical Carcinoma,” Proceedings of the National Academy of Sciences of the United States of America, 93(13):6704-9 (1996)). Competitive and real-time PCR are considerably faster and require less material than Southern hybridization, although neither technique is amenable to multiplexing. Current multiplex microsatellite marker approaches require careful attention to primer concentrations and amplification conditions. While PCR products may be pooled in sets, this requires an initial run on agarose gels to approximate the amount of DNA in each band (Reed, P. W., et al., “Chromosome-Specific Microsatellite Sets For Fluorescence-Based, Semi-Automated Genome Mapping,” Nat Genet, 7(3): 390-5 (1994), and Hampton, G. M., et al., “Simultaneous Assessment Of Loss Of Heterozygosity At Multiple Microsatellite Loci Using Semi-Automated Fluorescence-Based Detection: Subregional Mapping Of Chromosome 4 In Cervical Carcinoma,” Proc. Nat'l. Acad. Sci. USA, 93(13):6704-9 (1996)). CGH provides a global assessment of LOH and amplification, but with a resolution range of about 20 Mb. To improve gene mapping and discovery, new techniques are urgently needed to allow for simultaneous detection of multiple genetic alterations.
Amplified fragment length polymorphism (“AFLP”) technology is a powerful DNA fingerprinting technique originally developed to identify plant polymorphisms in genomic DNA. It is based on the selective amplification of restriction fragments from a total digest of genomic DNA.
The original technique involved three steps: (1) restriction of the genomic DNA, i.e. with EcoRI and MseI, and ligation of oligonucleotide adapters, (2) selective amplification of a subset of all the fragments in the total digest using primers which reached in by from 1 to 3 bases, and (3) gel-based analysis of the amplified fragments. Janssen, et al., “Evaluation of the DNA Fingerprinting Method AFLP as an New Tool in Bacterial Taxonomy,” Microbiology, 142(Pt 7):1881-93 (1996); Thomas, et al., “Identification of Amplified Restriction Fragment Polymorphism (AFLP) Markers Tightly Linked to the Tomato Cf-9 Gene for Resistance to Cladosporium fulvum,”. Plant J, 8(5):785-94 (1995); Vos, et al., “AFLP: A New Technique for DNA Fingerprinting,” Nucleic Acids Res, 23(21):4407-14 (1995); Bachem, et al., “Visualization of Differential Gene Expression Using a Novel Method of RNA Fingerprinting Based on AFLP: Analysis of Gene Expression During Potato Tuber Development,” Plant J, 9(5):745-53 (1996); and Meksem, et al., “A High-Resolution Map of the Vicinity of the R1 Locus on Chromosome V of Potato Based on RFLP and AFLP Markers,” Mol Gen Genet, 249(1):74-81 (1995), which are hereby incorporated by reference.
AFLP differs substantially from the present invention because it: (i) uses palindromic enzymes, (ii) amplifies both desired EcoRI-MseI as well as unwanted MseI-MseI fragments, and (iii) does not identify both alleles when a SNP destroys a pre-existing restriction site. Further, AFLP does not identify SNPs which are outside restriction sites. AFLP does not, and was not designed to create a map of a genome.
Representational Difference Analysis (RDA) was developed by N. Lisitsyn and M. Wigler to isolate the differences between two genomes (Lisitsyn, et al., “Cloning the Differences Between Two Complex Genomes,” Science, 259:946-951 (1993), Lisitsyn, et al., “Direct Isolation of Polymorphic Markers Linked to a Trait by Genetically Directed Representational Difference Analysis,” Nat Genet, 6(1):57-63 (1994); Lisitsyn, et al., “Comparative Genomic Analysis of Tumors: Detection of DNA Losses and Amplification,” Proc Natl Acad Sci USA, 92(1):151-5 (1995); Thiagalingam, et al., “Evaluation of the FHIT Gene in Colorectal Cancers,” Cancer Res, 56(13):2936-9 (1996), Li, et al., “PTEN, a Putative Protein Tyrosine Phosphatase Gene Mutated in Human Brain, Breast, and Prostate Cancer,” Science, 275(5308):1943-7 (1997); and Schutte, et al., “Identification by Representational Difference Analysis of a Homozygous Deletion in Pancreatic Carcinoma That Lies Within the BRCA2 Region,” Proc Natl Acad Sci USA, 92(13):5950-4 (1995). The system was developed in which subtractive and kinetic enrichment was used to purify restriction endonuclease fragments present in one DNA sample, but not in another. The representational part is required to reduce the complexity of the DNA and generates “amplicons”. This allows isolation of probes that detect viral sequences in human DNA, polymorphisms, loss of heterozygosities, gene amplifications, and genome rearrangements.
The principle is to subtract “tester” amplicons from an excess of “driver” amplicons. When the tester DNA is tumor DNA and the driver is normal DNA, one isolates gene amplifications. When the tester DNA is normal DNA and the driver is tumor DNA, one isolates genes which lose function (i.e. tumor suppressor genes).
A brief outline of the procedure is provided herein: (i) cleave both tester and driver DNA with the same restriction endonuclease, (ii) ligate unphosphorylated adapters to tester DNA, (iii) mix a 10-fold excess of driver to tester DNA, melt and hybridize, (iv) fill in ends, (v) add primer and PCR amplify, (vi) digest ssDNA with mung bean nuclease, (vii) PCR amplify, (viii) repeat steps (i) to (vii) for 2-3 rounds, (ix) clone fragments and sequence.
RDA differs substantially from the present invention because it: (i) is a very complex procedure, (ii) is used to identify only a few differences between a tester and driver sample, and (iii) does not identify both alleles when a SNP destroys a pre-existing restriction site. Further, RDA does not identify SNPs which are outside restriction sites. RDA does not, and was not designed to create a map of a genome.
The advent of DNA arrays has resulted in a paradigm shift in detecting vast numbers of sequence variation and gene expression levels on a genomic scale (Pease, A. C., et al., “Light-Generated Oligonucleotide Arrays For Rapid DNA Sequence Analysis,” Proc Natl Acad Sci USA, 91(11):5022-6 (1994), Lipshutz, R. J., et al., “Using Oligonucleotide Probe Arrays To Access Genetic Diversity,” Biotechniques, 19(3):442-7 (1995), Eggers, M., et al., “A Microchip For Quantitative Detection Of Molecules Utilizing Luminescent And Radioisotope Reporter Groups,” Biotechniques, 17(3):516-25 (1994), Guo, Z., et al., “Direct Fluorescence Analysis Of Genetic Polymorphisms By Hybridization With Oligonucleotide Arrays On Glass Supports,” Nucleic Acids Res, 22(24):5456-65 (1994), Beattie, K. L., et al., “Advances In Genosensor Research,” Clinical Chemistry, 41(5):700-6 (1995), Hacia, J. G., et al., “Detection Of Heterozygous Mutations In BRCA1 Using High Density Oligonucleotide Arrays And Two-Colour Fluorescence Analysis,” Nature Genetics, 14(4):441-7 (1996), Chee, M., et al., “Accessing Genetic Information With High-Density DNA Arrays,” Science, 274(5287):610-4 (1996), Cronin, M. T., et al., “Cystic Fibrosis Mutation Detection By Hybridization To Light-Generated DNA Probe Arrays,” Hum Mutat, 7(3):244-55 (1996), Drobyshev, A., et al., “Sequence Analysis By Hybridization With Oligonucleotide Microchip: Identification Of Beta-Thalassemia Mutations,” Gene, 188(1):45-52 (1997), Kozal, M. J., et al., “Extensive Polymorphisms Observed In HIV-1 Clade B Protease Gene Using High-Density Oligonucleotide Arrays,” Nature Medicine, 2(7):753-9 (1996), Yershov, G., et al., “DNA Analysis And Diagnostics On Oligonucleotide Microchips,” Proc Natl Acad Sci USA, 93(10):4913-8 (1996), DeRisi, J., et al., “Use Of A CDNA Microarray To Analyse Gene Expression Patterns In Human Cancer,” Nature Genetics, 14(4):457-60 (1996), Schena, M., et al., “Parallel Human Genome Analysis: Microarray-Based Expression Monitoring Of 1000 Genes,” Proc. Nat'l. Acad. Sci. USA, 93(20):10614-9 (1996), Shalon, D., et al., “A DNA Microarray System For Analyzing Complex DNA Samples Using Two-Color Fluorescent Probe Hybridization,” Genome Research, 6(7):639-45 (1996)). Determining deletions, amplifications, and mutations at the DNA level will complement the information obtained from expression profiling of tumors (DeRisi, J., et al., “Use Of A cDNA Microarray To Analyse Gene Expression Patterns In Human Cancer,” Nature Genetics, 14(4):457-60 (1996), and Zhang, L., et al., “Gene Expression Profiles In Normal And Cancer Cells,” Science, 276:1268-1272 (1997)). DNA chips designed to distinguish single nucleotide differences are generally based on the principle of “sequencing by hybridization” (Lipshutz, R. J., et al., “Using Oligonucleotide Probe Arrays To Access Genetic Diversity,” Biotechniques, 19(3):442-7 (1995), Eggers, M., et al., “A Microchip For Quantitative Detection Of Molecules Utilizing Luminescent And Radioisotope Reporter Groups,” Biotechniques, 17(3):516-25 (1994), Guo, Z., et al., “Direct Fluorescence Analysis Of Genetic Polymorphisms By Hybridization With Oligonucleotide Arrays On Glass Supports,” Nucleic Acids Res, 22(24):5456-65 (1994), Beattie, K. L., et al., “Advances In Genosensor Research,” Clinical Chemistry, 41(5):700-6 (1995), Hacia, J. G., et al., “Detection Of Heterozygous Mutations In BRCA1 Using High Density Oligonucleotide Arrays And Two-Colour Fluorescence Analysis,” Nature Genetics, 14(4):441-7 (1996), Chee, M., et al., “Accessing Genetic Information With High-Density DNA Arrays,” Science, 274(5287):610-4 (1996), Cronin, M. T., et al., “Cystic Fibrosis Mutation Detection By Hybridization To Light-Generated DNA Probe Arrays,” Hum Mutat, 7(3):244-55 (1996), Drobyshev, A., et al., “Sequence Analysis By Hybridization With Oligonucleotide Microchip: Identification Of Beta-Thalassemia Mutations,” Gene, 188(1):45-52 (1997), Kozal, M. J., et al., “Extensive Polymorphisms Observed In HIV-1 Clade B Protease Gene Using High-Density Oligonucleotide Arrays,” Nature Medicine, 2(7):753-9 (1996), and Yershov, G., et al., “DNA Analysis And Diagnostics On Oligonucleotide Microchips,” Proc Natl Acad Sci USA, 93(10):4913-8 (1996)), or polymerase extension of arrayed primers (Nikiforov, T. T., et al., “Genetic Bit Analysis: A Solid Phase Method For Typing Single Nucleotide Polymorphisms,” Nucleic Acids Research, 22(20):4167-75 (1994), Shumaker, J. M., et al., “Mutation Detection By Solid Phase Primer Extension,” Human Mutation, 7(4):346-54 (1996), Pastinen, T., et al., “Minisequencing: A Specific Tool For DNA Analysis And Diagnostics On Oligonucleotide Arrays,” Genome Research, 7(6):606-14 (1997), and Lockley, A. K., et al., “Colorimetric Detection Of Immobilised PCR Products Generated On A Solid Support,” Nucleic Acids Research, 25(6):1313-4 (1997) (See Table 2)). While DNA chips can confirm a known sequence, similar hybridization profiles create ambiguities in distinguishing heterozygous from homozygous alleles (Eggers, M., et al., “A Microchip For Quantitative Detection Of Molecules Utilizing Luminescent And Radioisotope Reporter Groups,” Biotechniques, 17(3):516-25 (1994), Beattie, K. L., et al., “Advances In Genosensor Research,” Clinical Chemistry, 41(5):700-6 (1995), Chee, M., et al., “Accessing Genetic Information With High-Density DNA Arrays,” Science, 274(5287):610-4 (1996), Kozal, M. J., et al., “Extensive Polymorphisms Observed In HIV-1 Clade B Protease Gene Using High-Density Oligonucleotide Arrays,” Nature Medicine, 2(7):753-9 (1996), and Southern, E. M., “DNA Chips: Analysing Sequence By Hybridization To Oligonucleotides On A Large Scale,” Trends in Genetics, 12(3):110-5 (1996)). Attempts to overcome this problem include using two-color fluorescence analysis (Hacia, J. G., et al., “Detection Of Heterozygous Mutations In BRCA1 Using High Density Oligonucleotide Arrays And Two-Colour Fluorescence Analysis,” Nature Genetics, 14(4):441-7 (1996)), 40 overlapping addresses for each known polymorphism (Cronin, M. T., et al., “Cystic Fibrosis Mutation Detection By Hybridization To Light-Generated DNA Probe Arrays,” Hum Mutat, 7(3):244-55 (1996)), nucleotide analogues in the array sequence (Guo, Z., et al., “Enhanced Discrimination Of Single Nucleotide Polymorphisms By Artificial Mismatch Hybridization,” Nature Biotech., 15:331-335 (1997)), or adjacent co-hybridized oligonucleotides (Drobyshev, A., et al., “Sequence Analysis By Hybridization With Oligonucleotide Microchip: Identification Of Beta-Thalassemia Mutations,” Gene, 188(1):45-52 (1997) and Yershov, G., et al., “DNA Analysis And Diagnostics On Oligonucleotide Microchips,” Proc Natl Acad Sci USA, 93(10):4913-8 (1996)). In a side-by-side comparison, nucleotide discrimination using the hybridization chips fared an order of magnitude worse than using primer extension (Pastinen, T., et al., “Minisequencing: A Specific Tool For DNA Analysis And Diagnostics On Oligonucleotide Arrays,” Genome Research, 7(6):606-14 (1997)). Nevertheless, solid phase primer extension also generates false positive signals from mononucleotide repeat sequences, template-dependent errors, and template-independent errors (Nikiforov, T. T., et al., “Genetic Bit Analysis: A Solid Phase Method For Typing Single Nucleotide Polymorphisms,” Nucl. Acids Res., 22(20):4167-75 (1994) and Shumaker, J. M., et al., “Mutation Detection By Solid Phase Primer Extension,” Human Mutation, 7(4):346-54 (1996)).
Over the past few years, an alternate strategy in DNA array design has been pursued. Combined with solution-based polymerase chain reaction/ligase detection assay (PCR/LDR) this array allows for accurate quantification of each SNP allele (See Table 2).
TABLE 2Comparison of high-throughput techniques to quantify known SNPs in clinical samples.TechniqueAdvantagesDisadvantagesHybridization on1) High density: up to 135,000 addresses.1) Specificity determined by hybridizationDNA array2) Scan for SNPs in thousands of loci.difficult to distinguish all SNPs.3) Detects small insertions/deletionsdifficult to quantify allelic imbalance2) Each new DNA target requires a new arrayMini-sequencing1) Uses high fidelity polymerase extension1) Cannot detect small insertions/deletions.(SNuPE) onminimizes false positive signal.2) Each new DNA target requires a new array.DNA array2) Potential for single-tube assayPCR/LDR with1) Uses high fidelity thermostable ligase;1) Requires synthesis of many ligation primerszip-code captureminimizes false positive signal.on universal2) Separates SNP identification from signal capture;DNA arrayavoids problems of false hybridization3) Quantify gene amplifications and deletions.4) Universal array works for all gene targets.For high throughput detection of specific multiplexed LDR products, unique addressable array-specific sequences on the LDR probes guide each LDR product to a designated address on a DNA array, analogous to molecular tags developed for bacterial and yeast genetics genetics (Hensel, M., et al., “Simultaneous Identification Of Bacterial Virulence Genes By Negative Selection,” Science, 269(5222):400-3 (1995) and Shoemaker, D. et al., “Quantitative Phenotypic Analysis Of Yeast Deletion Mutants Using A Highly Parallel Molecular Bar-Coding Strategy,” Nat Genet, 14(4):450-6 (1996)). The specificity of this reaction is determined by a thermostable ligase which allows detection of (i) dozens to hundreds of polymorphisms in a single-tube multiplex format, (ii) small insertions and deletions in repeat sequences, and (iii) low level polymorphisms in a background of normal DNA. By uncoupling polymorphism identification from hybridization, each step may be optimized independently, thus allowing for quantitative assessment of allele imbalance even in the presence of stromal cell contamination. This approach has the potential to rapidly identify multiple gene deletions and amplifications associated with tumor progression, as well as lead to the discovery of new oncogenes and tumor suppressor genes. Further, the ability to score hundreds to thousands of SNPs has utility in linkage studies (Nickerson, D. A., et al., “Identification Of Clusters Of Biallelic Polymorphic Sequence-Tagged Sites (pSTSs) That Generate Highly Informative And Automatable Markers For Genetic Linkage Mapping,” Genomics, 12(2):377-87 (1992), Lin, Z., et al., “Multiplex Genotype Determination At A Large Number Of Gene Loci,” Proc Natl Acad Sci USA, 93(6):2582-7 (1996), Fanning, G. C., et al., “Polymerase Chain Reaction Haplotyping Using 3′ Mismatches In The Forward And Reverse Primers: Application To The Biallelic Polymorphisms Of Tumor Necrosis Factor And Lymphotoxin Alpha,” Tissue Antigens, 50(1):23-31 (1997), and Kruglyak, L., “The Use of a Genetic Map of Biallelic Markers in Linkage Studies,” Nature Genetics, 17:21-24 (1997)), human identification (Delahunty, C., et al., “Testing The Feasibility Of DNA Typing For Human Identification By PCR And An Oligonucleotide Ligation Assay,” Am. J. Hum. Gen., 58(6):1239-46 (1996) and Belgrader, P., et al., “A Multiplex PCR-Ligase Detection Reaction Assay For Human Identity Testing,” Gen. Sci. & Tech., 1:77-87 (1996)), and mapping complex human diseases using association studies where SNPs are identical by decent (Collins, F. S., “Positional Cloning Moves From Perditional To Traditional,” Nat Genet, 9(4):347-50 (1995), Lander, E. S., “The New Genomics: Global Views Of Biology,” Science, 274(5287):536-9 (1996), Risch, N. et al., “The Future Of Genetic Studies Of Complex Human Diseases,” Science, 273(5281):1516-7 (1996), Cheung, V. G. et al., “Genomic Mismatch Scanning Identifies Human Genomic DNA Shared Identical By Descent,” Genomics, 47(1):1-6 (1998), Heung, V. G., et al., “Linkage-Disequilibrium Mapping Without Genotyping,” Nat Genet, 18(3):225-230 (1998), and McAllister, L., et al., “Enrichment For Loci Identical-By-Descent Between Pairs Of Mouse Or Human Genomes By Genomic Mismatch Scanning,” Genomics, 47(1):7-11 (1998)).
For 85% of epithelial cancers, loss of heterozygosity and gene amplification are the most frequently observed changes which inactivate the tumor suppressor genes and activate the oncogenes. Southern hybridizations, competitive PCR, real time PCR, microsatellite marker analysis, and comparative genome hybridization (CGH) have all been used to quantify changes in chromosome copy number (Ried, T., et al., “Comparative Genomic Hybridization Reveals A Specific Pattern Of Chromosomal Gains And Losses During The Genesis Of Colorectal Tumors,” Genes, Chromosomes & Cancer, 15(4):234-45 (1996), Kallioniemi, et al., “ERBB2 Amplification In Breast Cancer Analyzed By Fluorescence In Situ Hybridization,” Proc Natl Acad Sci USA, 89(12):5321-5 (1992), Kallioniemi, et al., “Comparative Genomic Hybridization: A Rapid New Method For Detecting And Mapping DNA Amplification In Tumors,” Semin Cancer Biol, 4(1):41-6 (1993), Kallioniemi, et al., “Detection And Mapping Of Amplified DNA Sequences In Breast Cancer By Comparative Genomic Hybridization,” Proc Natl Acad Sci USA, 91(6):2156-60 (1994), Kallioniemi, et al., “Identification Of Gains And Losses Of DNA Sequences In Primary Bladder Cancer By Comparative Genomic Hybridization,” Genes Chromosom Cancer, 12(3):213-9 (1995), Schwab, M., et al., “Amplified DNA With Limited Homology To Myc Cellular Oncogene Is Shared By Human Neuroblastoma Cell Lines And A Neuroblastoma Tumour,” Nature, 305(5931):245-8 (1983), Solomon, E., et al., “Chromosome 5 Allele Loss In Human Colorectal Carcinomas,” Nature, 328(6131):616-9 (1987), Law, D. J., et al., “Concerted Nonsyntenic Allelic Loss In Human Colorectal Carcinoma,” Science, 241(4868):961-5 (1988), Frye, R. A., et al., “Detection Of Amplified Oncogenes By Differential Polymerase Chain Reaction,” Oncogene, 4(9):1153-7 (1989), Neubauer, A., et al., “Analysis Of Gene Amplification In Archival Tissue By Differential Polymerase Chain Reaction,” Oncogene, 7(5):1019-25 (1992), Chiang, P. W., et al., “Use Of A Fluorescent-PCR Reaction To Detect Genomic Sequence Copy Number And Transcriptional Abundance,” Genome Research, 6(10):1013-26 (1996), Heid, C. A., et al., “Real Time Quantitative PCR,” Genome Research, 6(10):986-94 (1996), Lee, H. H., et al., “Rapid Detection Of Trisomy 21 By Homologous Gene Quantitative PCR (HGQ-PCR),” Human Genetics, 99(3):364-7 (1997), Boland, C. R., et al., “Microallelotyping Defines The Sequence And Tempo Of Allelic Losses At Tumour Suppressor Gene Loci During Colorectal Cancer Progression,” Nature Medicine, 1(9):902-9 (1995), Cawkwell, L., et al., “Frequency Of Allele Loss Of DCC, p53, RBI, WT1, NF1, NM23 And APC/MCC In Colorectal Cancer Assayed By Fluorescent Multiplex Polymerase Chain Reaction,” Br J Cancer, 70(5):813-8 (1994), and Hampton, G. M., et al., “Simultaneous Assessment Of Loss Of Heterozygosity At Multiple Microsatellite Loci Using Semi-Automated Fluorescence-Based Detection: Subregional Mapping Of Chromosome 4 In Cervical Carcinoma,” Proc. Nat'l. Acad. Sci. USA, 93(13):6704-9 (1996)). Recently, a microarray of consecutive BACs from the long arm of chromosome 20 has been used to accurately quantify 5 regions of amplification and one region of LOH associated with development of breast cancer. This area was previously thought to contain only 3 regions of amplification (Tanner, M. et al., “Independent Amplification And Frequent Co-Amplification Of Three Nonsyntenic Regions On The Long Arm Of Chromosome 20 In Human Breast Cancer,” Cancer Research, 56(15):3441-5 (1996)). Although this approach will yield valuable information from cell lines, it is not clear it will prove quantitative when starting with microdissected tissue which require PCR amplification. Competitive and real time PCR approaches require careful optimization to detect 2-fold differences (Frye, R. A., et al., “Detection Of Amplified Oncogenes By Differential Polymerase Chain Reaction,” Oncogene, 4(9):1153-7 (1989), Neubauer, A., et al., “Analysis Of Gene Amplification In Archival Tissue By Differential Polymerase Chain Reaction,” Oncogene, 7(5):1019-25 (1992), Chiang, P. W., et al., “Use Of A Fluorescent-PCR Reaction To Detect Genomic Sequence Copy Number And Transcriptional Abundance,” Genome Research, 6(10):1013-26 (1996), Heid, C. A., et al., “Real Time Quantitative PCR,” Genome Research, 6(10):986-94 (1996), and Lee, H. H., et al., “Rapid Detection Of Trisomy 21 By Homologous Gene Quantitative PCR (HGQ-PCR),” Human Genetics, 99(3):364-7 (1997)). Unfortunately, stromal contamination may reduce the ratio between tumor and normal chromosome copy number to less than 2-fold. By using a quantitative SNP-DNA array detection, each allele can be distinguished independently, thus reducing the effect of stromal contamination in half. Further by comparing the ratio of allele-specific LDR product formed from a tumor to control gene between a tumor and normal sample, it may be possible to distinguish gene amplification from loss of heterozygosity at multiple loci in a single reaction.
Using PCR/LDR to Detect SNPs.
The ligase detection reaction (“LDR”) is ideal for multiplexed discrimination of single-base mutations or polymorphisms (Barany, F., et al., “Cloning, Overexpression, And Nucleotide Sequence Of A Thermostable DNA Ligase Gene,” Gene, 109:1-11 (1991), Barany, F., “Genetic Disease Detection And DNA Amplification Using Cloned Thermostable Ligase,” Proc. Natl. Acad. Sci. USA, 88:189-193 (1991), and Barany, F., “The Ligase Chain Reaction (LCR) In A PCR World,” PCR Methods and Applications, 1:5-16 (1991)). Since there is no polymerization step, several probe sets can ligate along a gene without interference. The optimal multiplex detection scheme involves a primary PCR amplification, followed by either LDR (two probes, same strand) or ligase chain reaction (“LCR”) (four probes, both strands) detection. This approach has been successfully applied for simultaneous multiplex detection of 61 cystic fibrosis alleles (Grossman, P. D., et al., “High-Density Multiplex Detection Of Nucleic Acid Sequences: Oligonucleotide Ligation Assay And Sequence-Coded Separation,” Nucleic Acids Res., 22:4527-4534 (1994) and Eggerding, F. A., et al., “Fluorescence-Based Oligonucleotide Ligation Assay For Analysis Of Cystic Fibrosis Transmembrane Conductance Regulator Gene Mutations,” Human Mutation, 5:153-165 (1995)), 6 hyperkalemic periodic paralysis alleles (Feero, W. T., et al., “Hyperkalemic Periodic Paralysis: Rapid Molecular Diagnosis And Relationship Of Genotype To Phenotype In 12 Families,” Neurology, 43:668-673 (1993)), and 20 21-hydroxylase deficiency alleles (Day, D., et al., “Detection Of Steroid 21 Hydroxylase Alleles Using Gene-Specific PCR And A Multiplexed Ligation Detection Reaction,” Genomics, 29:152-162 (1995) and Day, D. J., et al., “Identification Of Non-Amplifying CYP21 Genes When Using PCR-Based Diagnosis Of 21-Hydroxylase Deficiency In Congenital Adrenal Hyperplasia (CAH) Affected Pedigrees,” Hum Mol Genet, 5(12):2039-48 (1996)).
21-hydroxylase deficiency has the highest carrier rate of any genetic disease, with 6% of Ashkenazi Jews being carriers. Approximately 95% of mutations causing 21-hydroxylase deficiency are the result of recombinations between an inactive pseudogene termed CYP21P and the normally active gene termed CYP21, which share 98% sequence homology (White, P. C., et al., “Structure Of Human Steroid 21-Hydroxylase Genes,” Proc. Natl. Acad. Sci. USA, 83:5111-5115 (1986)). PCR/LDR was developed to rapidly determine heterozygosity or homozygosity for any of the 10 common apparent gene conversions in CYP21. By using allele-specific PCR, defined regions of CYP21 are amplified without amplifying the CYP21P sequence. The presence of wild-type or pseudogene mutation is subsequently determined by fluorescent LDR. Discriminating oligonucleotides complementary to both CYP21 and CYP21P are included in equimolar amounts in a single reaction tube so that a signal for either active gene, pseudogene, or both is always obtained. PCR/LDR genotyping (of 82 samples) was able to readily type compound heterozygotes with multiple gene conversions in a multiplexed reaction, and was in complete agreement with direct sequencing/ASO analysis. This method was able to distinguish insertion of a single T nucleotide into a (T)7 tract, which cannot be achieved by allele-specific PCR alone (Day, D., et al., “Detection Of Steroid 21 Hydroxylase Alleles Using Gene-Specific PCR And A Multiplexed Ligation Detection Reaction,” Genomics, 29:152-162 (1995)). A combination of PCR/LDR and microsatellite analysis revealed some unusual cases of PCR allele dropout (Day, D. J., et al., “Identification Of Non-Amplifying CYP21 Genes When Using PCR-Based Diagnosis Of 21-Hydroxylase Deficiency In Congenital Adrenal Hyperplasia (CAH) Affected Pedigrees,” Hum Mol Genet, 5(12):2039-48 (1996)). The LDR approach is a single-tube reaction which enables multiple samples to be analyzed on a single polyacrylamide gel.
A PCR/LDR assay has been developed to detect germline mutations, found at high frequency (3% total), in BRCA1 and BRCA2 genes in the Jewish population. The mutations are: BRCA1, exon 2 185delAG; BRCA1, exon 20 5382insC; BRCA2, exon 11 6174delT. These mutations are more difficult to detect than most germline mutations, as they involve slippage in short repeat regions. A preliminary screening of 20 samples using multiplex PCR of three exons and LDR of six alleles in a single tube assay has successfully detected the three Ashkenazi BRCA1 and BRCA2 mutations.
Multiplexed PCR for Amplifying Many Regions of Chromosomal DNA Simultaneously.
A coupled multiplex PCR/PCR/LDR assay was developed to identify armed forces personnel. Several hundred SNPs in known genes with heterozygosities, >0.4 are currently listed. Twelve of these were amplified in a single PCR reaction as follows: Long PCR primers were designed to have gene-specific 3′ ends and 5′ ends complementary to one of two sets of PCR primers. The upstream primers were synthesized with either FAM- or TET-fluorescent labels. These 24 gene-specific primers were pooled and used at low concentration in a 15 cycle PCR. After this, the two sets of primers were added at higher concentrations and the PCR was continued for an additional 25 cycles. The products were separated on an automated ABD 373A DNA Sequencer. The use of these primers produces similar amounts of multiplexed products without the need to carefully adjust gene-specific primer concentrations or PCR conditions (Belgrader, P., et al., “A Multiplex PCR-Ligase Detection Reaction Assay For Human Identity Testing,” Genome Science and Technology, 1:77-87 (1996)). In a separate experiment, non-fluorescent PCR products were diluted into an LDR reaction containing 24 fluorescently labeled allele-specific LDR probes and 12 adjacent common LDR probes, with products separated on an automated DNA sequencer. LDR probe sets were designed in two ways: (i) allele-specific FAM- or TET-labeled LDR probes of uniform length, or (ii) allele-specific HEX-labeled LDR probes differing in length by two bases. A comparison of LDR profiles of several individuals demonstrated the ability of PCR/LDR to distinguish both homozygous and heterozygous genotypes at each locus (Id.). The use of PCR/PCR in human identification to simultaneously amplify 26 loci has been validated (Lin, Z., et al., “Multiplex Genotype Determination At A Large Number Of Gene Loci,” Proc Natl Acad Sci USA, 93(6):2582-7 (1996)), or ligase based detection to distinguish 32 alleles although the latter was in individual reactions (Nickerson, D. A., et al., “Identification Of Clusters Of Biallelic Polymorphic Sequence-Tagged Sites (pSTSs) That Generate Highly Informative And Automatable Markers For Genetic Linkage Mapping,” Genomics, 12(2):377-87 (1992)). This study validates the ability to multiplex both PCR and LDR reactions in a single tube, which is a prerequisite for developing a high throughput method to simultaneously detect SNPs throughout the genome.
For the PCR/PCR/LDR approach, two long PCR primers are required for each SNP analyzed. A method which reduces the need for multiple PCR primers would give significant savings in time and cost of a large-scale SNP analysis. The present invention is directed to achieving this objective.